摘要
背景
肝细胞癌(Hepatocellular carcinoma,HCC)是常见、高发的恶性肿瘤之一肝细胞癌术后的高复发率成为影响其生存率和预后的最主要因素之一。但其根本的病因及机制尚不明了。深入研究原发性肝癌发生、发展的分子生物学基础,及早预测原发性肝癌复发的危险性,从而有效改善原发性肝癌病人的预后,在当前有着非常重要的现实意义。核仁纺锤体相关蛋白1(nucleolar spindle-associated protein1,NuSAP1)是一种微管合蛋白,在纺锤体组装中发挥重要功能,是保证细胞周期正常进行的重要调控分子。NuSAP1的mRNA水平和蛋白水平受到严格的调控,在细胞周期进程中呈现周期性变化。转录因子E2F家族在控制细胞周期的进展中起关键作用,NuSAP1基因的启动子区域含有E2F的结合位点,E2F1和E2F结合位点结合后可明显促进NuSAP1的转录。近年来研究发现在许多其它肿瘤中存在NuSAP1的过表达,它们的表达水平往往和肿瘤的侵袭性及预后有关。有关NuSAP1在原发性肝癌发生发展中的作用以及与肿瘤侵袭性、预后的关系还没有相关的报道。
目的
鉴于NuSAP1家族在细胞周期中的重要作用,其功能的紊乱与肿瘤的发生发展及转移复发可能存在着密切关系,本实验将从细胞水平和组织水平研究NuSAP1在肝细胞癌中的表达,分析NuSAP1在肝细胞癌中表达的临床意义及其与E2F1的关系,探讨NuSAP1在肝细胞癌的发生、发展和复发转移过程中的作用及其分子机制。
方法
(I).收集手术切除的新鲜肝细胞癌组织和相应的非癌组织标本,采用RT-PCR,荧光定量PCR检测癌和非癌组织标本中NuSAP1的mRNA表达水平,免疫组织化学染色检测癌和非癌组织标本中NuSAP1、E2F1蛋白表达水平,分别分析NuSAP1、E2F1在肝细胞癌中的表达与相关临床病理特征及预后的关系；并探讨E2F1在肝细胞癌中的表达及其与NuSAP1表达的关系。
(11).采用人高转移肝细胞癌细胞株HCCLM3,利用SiRNA定向抑制NuSAP1的表达,采用荧光定量PCR方法检测抑制前后HCCLM3细胞株中NuSAP1mRNA表达的变化；采用Western blotting方法检测抑制前后HCCLM3细胞株中NuSAP1蛋白表达的变化；使用Freshney计数板计量细胞数目的变化；MTS和Edu法检测细胞增殖活性的变化；transwell小室检测细胞迁移能力和细胞侵袭能力的变化：流式细胞仪检测细胞周期和凋亡的变化；分析其表达对肝细胞癌细胞生物学特性的影响；
(III).采用人高转移肝细胞癌细胞株MHCC97H,利用SiRNA定向抑制E2F1的表达；采用荧光定量PCR方法检测抑制前后MHCC97H细胞株中NuSAP1mRNA表达的变化；采用Western blotting方法检测抑制前后MHCC97H细胞株中NuSAP1蛋白表达的变化；研究在肝细胞癌细胞株中E2F1的表达对NuSAP1表达的调节作用。
结果
1.无论是核酸水平还是蛋白水平,NuSAP1在肝细胞癌组织中的表达均明显高于相应的非癌组织(P<0.05)。单因素分析表明：NuSAP1mRNA和蛋白的表达均与TNM分期、巴塞罗那分期、淋巴结转移、早期复发、有无癌栓和病理分化相关(P<0.05)；NuSAP1mRNA的表达与血清AFP相关,而NuSAP1蛋白的表达与肿瘤大小、肝硬化相关(P<0.05)；多因素逐步logistic回归分析表明：NuSAP1蛋白的表达是早期复发的独立影响因素(P<0.05)。生存分析表明NuSAP1在肝细胞癌组织中的高表达与肝细胞癌患者预后差相关(X2=5.939,P=0.015)。肝细胞癌患者术后的无瘤生存期与NuSAP1蛋白表达的高低相关(Kaplan-Meier, log-rank test, P<0.05)。
2.E2F1蛋白在肝细胞癌组织中的表达明显升高(P<0.05)；肝细胞癌组织中的E2F1蛋白表达升高与病理分化、早期复发、TNM分期及巴塞罗那分期相关(P<0.05)；生存分析表明E2F1在肝细胞癌组织中的高表达也与肝细胞癌患者预后差相关(X2=8.952,P=0.003)。肝细胞癌患者术后的无瘤生存期也与E2F1蛋白表达的高低相关(Kaplan-Meier, log-rank test, P<0.05)。并且E2F1与NUSAP1蛋白的表达呈正相关的关系(P<0.05)；
3.在肝细胞癌细胞株的抑制实验中,抑制HCCLM3细胞株NuSAP1的表达可明显降低肝细胞癌的细胞数目的增加、抑制细胞增殖活性、细胞移动性及细胞侵袭性、促进G2/M期转化、促进细胞凋亡等(P<0.05)；抑制MHCC97H细胞株E2F1的表达可明显降低NuSAP1的表达量(P<0.05)。
结论
1、NuSAP1在肝细胞癌组织中存在过表达,NuSAP1在肝细胞癌中的表达与肝细胞癌患者的早期复发有关,NuSAP1高表达者预后差。
2、E2F1在肝细胞癌组织中的表达明显升高,并且与NuSAP1的表达存在正相关,影响肝细胞癌患者的预后。
3、NuSAP1表达在肝细胞癌中可能起癌基因的作用：NuSAP1可能作为癌基因参与肝细胞癌的发生和发展。
4、肝细胞癌细胞中NuSAP1的表达上调可能是通过E2F1介导的。
[Backgrounds]
Hepatocellular carcinoma (HCC) is one of the most common and frequent malignancies. Its high rate of recurrence after surgical resection is one of the key reasons for the low survival rates and a poor prognosis. However, the cause and the machenism of HCC remains unexplored. To improve patient prognosis, it is very important to elucidate the biological mechanisms that control the tumor-initiating and development and predict the early recurrence of HCC. Nucleolar-spindle associated protein1(NuSAP1) is a microtubule-associated protein that plays a central role in spindle assembly and act as a key regulator to warrant the process of cell division.The levels of NuSAP1expression were strictly regulated and dynamically changed during different stages of the cell cycle. The transcription factor E2F family play a key roles in cotrolling the cell cycle. The binging of E2F1to its binding site which located on the promoter of NuSAP1was able to markedly promote the NuSAP1transcripton. Recent studies indentify overexpression of NuSAP1in many other tumors,and the expression level of NuSAP1seem to be related to the invasiveness and prognosis of the tumors.But whether the same mechanism exsit in HCC has not been report.
[OBJECTIVE]
Owing to the important function of the NuSAP1in the cell cycle,the disturb of its function may associated with the development and recurrence of cancer. The aims of the research are mainly to investigate the expression level of NuSAP1in HCC cell lines and HCC tissues,elucidate its clinical significances and its relationship with E2F1,explore its role in occurrence, development and recurrence of hepatocellular carcinoma.
[METHODS]
(I).The expression level of NuSAP1and its mRNA, E2F1protein in tumor tissues and non-tumor tissues from patients with HCC was detected by semi-qRT-PCR, quantitative real-time PCR and immunohistochemical; the relationship between its expression and the clinicopathologic features was studied respectively. Then the correlations between NuSAP1and E2F1in HCC were studied.(Ⅱ). HCCLM3cell line with high aggressive phenotype was choose, After inhibited with siRNAs, NuSAP1mRNA and protein expression level were detected by quantitative real-time PCR and Western blotting.Cell proliferation activity was analysised by the method of MTS and Edu,Cell migration and aggressive capability was tested by the method of transwell.Flow cytometry was used to determined cell cycle and apoptosis, respectively.(Ⅲ). After inhibited the expression of E2F1in HMHCC97H cell line with siRNAs, NuSAP1mRNA and protein expression level were detected by quantitative real-time PCR and Western blotting(analysis among the inhibite-before and inhibite-after HCC cell lines),and then their relationship between E2F1and NuSAP1in hepatocellular carcinoma were studied.
[RESULTS]
1.There were apparently higher expressions of NuSAP1and its mRNA in HCC tumor tissues than in non-tumor tissues(P<0.05).With univariate analysis showed that the expression of NuSAP1and its mRNA in HCC was associated with TNM classification, BCLC classification, lymphatic metastasis, early recurrence, tumor thrombi and histological differentiation (P<0.05). Meal while, the expression of NuSAP1mRNA was also associated with the serum AFP level;The expression of NuSAP1was also associated with tumor size and liver cirrhosis.With multivariate analvsis, NuSAPl is a independent risk factor for early recurrence (P=0.005).Survival analysis showed that the expression of NuSAP1in HCC was associated with postoperative recurrence (X2=5.939, P=0.015). The surival time without tumor was positively correlated with NuSAP1protein expression (Kaplan-Meier, log-rank test, P<0.05).
2.The expressions of E2F1protein were apparently higher in tumor tissues than in non-tumor tissues(P<0.05). The expression of E2F1protein in HCC was associated with histological differentiation, early recurrence, BCLC classification and TNM classification (P<0.05), but not to sex, age, tumor thrombi, tumor size, liver cirrhosis, lymphatic metastasis,tumor number, AFP level,HBV and the presence of tumor encapsulation (P>0.05). Survival analysis showed that the expression of E2F1protein in HCC was associated with postoperative recurrence (X2=8.952, P=0.003). The surival time without tumor was positively correlated with E2F1protein expression (Kaplan-Meier, log-rank test, P<0.05). There was positive correlation between the expression of E2F1protein and NuSAPl in tumor tissues (P<0.05);
3.1n the study of inhibited HCC cell lines,inhibited the expression of NuSAP1could apparently decrease the cell numbers,cell proliferation activity,cell migration capability and cell aggressive capability,but increase the cell cycle G2/M transform and cell apoptosis(P<0.05); To inhibite the expression of E2F1protein could apparently decrease the expression of NuSAP1(P<0.05).
[CONCLUSION]
1、The expression of NuSAP1in HCC tissues was higher than that in non-tumor tissues, and correlated with postoperative early recurrence, Higher expression of NuSAP1might lead to a poor prognosis.
2, The expression of E2F1protein in HCC tissues was higher than that in non-tumor tissues, and positive correlated with the level of NuSAPl,this indicate E2F1protein maybe act as a regulator to NuSAP1.
3、NuSAPl maybe a oncogene in HCC cell lines.,its high expression maybe take part in HCC angiogenesis and development.
4、E2F1maybe act as a regulator to NuSAPl in HCC cell lines.
引文
[1]Parkin DM, Bray F, Ferlay J, et al. Global cancer statistics[J],2002, CA Cancer J Clin,2005,55(2):74-108.
[2]Villanueva A, Newell P, Chiang DY, et al. Genomics and signaling pathways in hepatocellular carcinoma[J]. Semin Liver Dis,2007,27(1):55-76.
[3]Schwartz GK, Shah MA. Targeting the cell cycle:a new approach to cancer therapy[J]. J Clin Oncol,2005,23(36):9408-9412.
[4]Nogales E. Structural insight into microtubule function[J]. Annu Rev Biophys Biomol Struct,2001,30:397-420.
[5]Andersen SS. Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18[J]. Trends Cell Biol,2001,10(7):261-267.
[6]Raemaekers T, Ribbeck K, Beandouin J,et al. NuSAP1, a novel microtubule. associated protein involved in mitotic spindle organization[J]. J Cell Biol.2003,162(6):1017-1029.
[7]Ribbeek K, Green AC, Santarelia R, et al. NuSAP1, a mitotic RanGTP target that stabilizes and cross-links microtubules[J]. Mol Biol Cell.2006,17(6): 2646-2660.
[8]Song L, Rape M. Regulated degradation of spindle assembly factors by the anaphase—promoting complex[J].Mol Cell,2010,14(3):369-382.
[9]Vanden BA, Raemaekers T, Denayer S,et al. NuSAP1 is essential for chromatin. induced spindle formation during early embryngenesis [J]. J Cell Sci,2010.123(19):3244-3255.
[10]Iaquinta PJ, Lees JA. Life and death decisions by the E2F transcription factors[J]. Curr Opin Cell Biol,2007,19(6):649-617.
[11]Verdaguer E, Susana Gde A, Clemens A,et al. Implication of the transcription factor E2F-1 in the modulation of neuronal apoptosis [J]. Biomed Pharmacother, 2007,61(7):390-399.
[12]DeGregori J, Johnson DG Distinct and overlapping roles for E2F family members in transcription, proliferation and apoptosis[J]. Curr Mol Med,2006, 6(7):739-748.
[13]Maiti B, Li J, de Bruin A, et al. Cloning and characterization of mouse E2F8, a novel mammalian E2F family member capable of blocking cellular proliferation [J]. J Biol Chem,2005,280 (18):18211-18220.
[14]Iwamoto M, Banerjee D, Menon LQ et al. Overexpression of E2F-1 in lung and liver metastases of human colon cancer is associated with gene amplification[J]. Cancer Biol Ther,2004,3(4):395-399.
[15]Canepa ET, Scassa ME, Ceruti JM, et al. INK4 proteins, a family of mammalian CDK inhibitors with novel biological functions [J].IUBMB Life, 2007,59(7):419-426.
[16]Gulzar ZG,McKenney JK, Brooks JD. Increased expression of NuSAPl in recurrent prostate cancer is mediated by E2F1 [J]. Oncogene.2013,32(1):70-77.
[17]Iyer J, Moghe S, Furukawa M, et al. What's Nu(SAP) in mitosis and cancer[J]? Cell Signal.2011,23(6):991-8.
[18]Satow R, Shitashige M, Kanai Y,et al. Combined functional genome survey of therapeutic targets for hepatocellular carcinoma. Clin Cancer Res. 2010.16(9):2518-28.
[19]Marsh JW, Dvorchik I, Bonham CA,et al. Is the pathologic TNM staging system for patients with hepatoma predictive of outcome? Cancer 2000; 88: 538-543.
[20]Marrero JA, Kudo M, Bronowicki JP. The challenge of prognosis and staging for hepatocellular carcinoma. Oncologist.2010;15:23-33.
[21]Scrensen JB, Klee M, Palshof T, et al. Performance status assessment in cancer patients. An inter-observer variability study. Br J Cancer 1993;67(4):773-775.
[22]卢欣,赵海涛,毛一雷,等.肝细胞肝细胞癌患者术后早期复发情况[J].中国医学科学院学报,2008,30(4),415—420.
[23]谢萍,李璐,张令强,等.微管结合蛋白NuSAP1的组织表达谱分析.军事 医学科学院院刊,2010,34(6)：509-512.
[24]许良中。杨文涛.免疫组织化学反应结果的判断标准.中国癌症杂志,1996,6：229-31.
[25]Fujiwara T, Harigae H, Okitsu Y, et al. Expression analyses and transcriptional regulation of mouse nucleolar spindle-associated protein gene in erythroid cells: essential role of NF-Y[J].Br J Haematol.2006 Nov;135(4):583-90.
[26]Fujiwara T, Harigae H, Takahashi S, et al. Differential gene expression profiling between wild-type and ALAS2-null erythroblasts:identification of novel heme-regulated genes.Biochem Biophys Res Commun[J].2006 340(1):105-10.
[27]Marie SK, Okamoto OK, Uno M, et al. Maternal embryonic leucine zipper kinase transcript abundance correlates with malignancy grade in human astrocytomas[J].Int J Cancer.2008,122(4):807-15.
[28]Ryu B, Kim DS, Deluca AM,et al. Comprehensive expression profiling of tumor cell lines identifies molecular signatures of melanoma progression[J].PLoS One.2007,2(7):e594.
[29]Bogunovic D, O'Neill DW, Belitskaya-Levy I, et al. Immune profile and mitotic index of metastatic melanoma lesions enhance clinical staging in predicting patient survival[J].Proc Natl Acad Sci U S A.2009,106(48):20429-34.
[30]Chen DT, Nasir A, Culhane A, et al. Proliferative genes dominate malignancy-risk gene signature in histologically-normal breast tissue [J].Breast Cancer Res Treat.2010,119(2):335-46.
[31]Lauss M, Kriegner A, Vierlinger K, et al. Consensus genes of the literature to predict breast cancer recurrence[J].Breast Cancer Res Treat.2008,110(2): 235-44.
[32]Nie J, Wang H, He F, et al. NuSAP1 is essential for neural crest cell migration in zebrafish. Protein Cell.2010,1(3):259-66.
[33]Recchia FM, Xu L, Penn JS, et al. Identification of genes and pathways involved in retinal neovascularization by microarray analysis of two animal models of retinal angiogenesis. Invest Ophthalmol Vis Sci.2010, 51(2):1098-105.
[34]Palaiologou M, Koskinas J, Karanikolas M, et al.E2F-1 is overexpressed and pro-apoptotic in human hepatocellular carcinoma[J].Virchows Arch, 2012,460(5):439-46.
[35]Kovesdi I, Reichel R, Nevins JR. Identification of a cellular transcription factor involved in E1A transactivation [J]. Cell,1986,45(2):219-228.
[36]Helfenstein A, Frahm SO, Krams M, et al.Minichromosome maintenance protein (MCM6) in low-grade chondrosarcoma:distinction from enchondroma and identification of progressive tumors [J].Am J Clin Pathol,2004,122:912-18.
[37]Lammers LA, Mathijssen RH, van Gelder T, et al.The impact of CYP2D6-predicted phenotype on tamoxifen treatment outcome in patients with metastatic breast cancer [J].Br J Cancer,2010,103:765-71.
[38]Miyata Y, Sakai H, Kanda S, et al. Expression of insulin-like growth factor binding protein-3 before and after neoadjuvant hormonal therapy in human prostate cancer tissues:correlation with histopathologic effects and biochemical recurrence[J]. Urology,2004,63:1184-90.
[39]Monge M, Colas E, Doll A, et al. Proteomic approach to ETV5 during endometrial carcinoma invasion reveals a link to oxidative stress[J]. Carcinogenesis,2009,30:1288-97.
[40]Tian J,Tang ZY,Ye SL,et al.New human hepatocellular carcinoma (HCC) cell line with highly metastatic potential (MHCC97H) and its expressions of the factors associated with metastasis[J].Br J Cancer,1999,81(5):814-21.
[41]Li Y,Tang ZY,Ye SL,et al.Establishment of cell clones with differrent metastatic potential from the metastatic hepatocellular carcinoma cell line MHCC97H[J].World J Gastroenterol,2001,7(5):630-6.
[42]Li Y,Tang Y,Ye L,et al.Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray[J].J Cancer Res Clin Oncol,2003,129(1):43-51.
[43]Ogawa H, Ishiguro K, Gaubatz S, et al. A complex with chromatin Ogawa H, Ishiguro K, Gaubatz S, et al. A complex with chromatin modifiers that occupies E2F and Myc-responsive genes in GO cells [J].Science,2002, 296(5570):1132-1136.
[1]Nogales E. Structural insight into microtubule function[J]. Annu Rev Biophys Biomol Struct,2001,30:397-420.
[2]Andersen SS. Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Opl8[J]. Trends Cell Biol,2001,10(7):261-267.
[3]Raemaekers T, Ribbeck K, Beandouin J,et al. NuSAP1, a novel microtubule. associated protein involved in mitotic spindle organization[J]. J Cell Biol.2003,162(6):1017-1029.
[4]Vanden BA, Raemaekers T, Denayer S,et al. NuSAPl is essential for chromatin. induced spindle formation during early embryngenesis [J]. J Cell Sci,2010.123(19):3244-3255.
[5]Iyer J, Moghe S, Furukawa M, et al. What's Nu(SAP) in mitosis and cancer[J]? Cell Signal.2011,23(6):991-8.
[6]L.A. Kelley, M.J.E. Sternberg, Nat. Protoc.4 (2009) 363.
[7]F. Kiefer, K. Arnold, M. Kunzli, L. Bordoli, T. Schwede, Nucleic Acids Res.37 (2009)D387.
[8]Raemaekers T, Ribbeck K, Beandouin J,et al. NuSAP1, a novel microtubule. associated protein involved in mitotic spindle organization[J]. J Cell Biol.2003,162(6):1017-29.
[9]Li L, Zhou Y, Sun L, et al. NuSAP1 is degraded by APC/C-Cdhl and its overexpression results in mitotic arrest dependent of its microtubules'affinity[J]. Cell signal,2007,19(10):2046-2055.
[10]L. Aravind, E.V. Koonin, Trends Biochem. Sci.25 (2000) 112.
[11]K. Ribbeck, T. Raemaekers, G.Carmeliet, I.W. Mattaj, Curr. Biol.17 (2007) 230.
[12]L. Song, M. Rape, Mol. Cell 38 (2010) 369.
[13]K. Ribbeck, A.C. Groen, R. Santarella, M.T. Boh nsack, T. Raemaeke rs, T. Kocher, M.Gentzel, D. Gorlich, M. Wilm, G Carmeliet, T.J. Mitchison, J. Ellenberg, A. Hoenger,I.W. Mattaj, Mol. Biol. Cell 17 (2006) 2646.
[14]S. Hussain, S.B. Benavente, E. Nascimento, I. Dragoni, A. Kurowski, A. Gillich, P.Humphreys, M. Frye, J. Cell Biol.186 (2009) 27.
[15]P.P. Wadia, M. Coram, R.J. Armstrong, M. Mindrinos, A.J. Butte, D.B. Miklos, Blood115 (2010) 2077.
[16]Gulzar ZG, McKenney JK, Brooks JD. Increased expression of NuSAP1 in recurrent prostate cancer is mediated by E2F1[J]. Oncogene.2012, doi: 10.1038.
[17]N. Reichert, S. Wurster, T. Ulrich, K. Schmitt, S. Hauser, L. Probst, R. Gotz, F. Ceteci,R. Moll, U. Rapp, S. Gaubatz, Mol. Cell. Biol.30 (2010) 2896.
[18]M.A. Kleinschmidt, T.U. Wagner, D. Liedtke, S. Spahr, B. Samans, S. Gaubatz, J. Biol.Chem.284 (2009) 13119.
[19]T. Fuj iwara, H. Harig ae, Y. Okitsu, S. Takahashi, H. Yokoyama, M.F. Yamada, K.Ishizawa, J. Kameoka, M. Kaku, T. Sasaki, Br. J. Haematol.135 (2006)583.
[20]M. Takano, Z. Lu, T. Goto, L. Fusi, J. Higham, J. Franc is, A. Withey, J. Hardt, B. Cloke,A.V. Stavropoulou, O. Ishihara, E.W.-. Lam, T.G Unter man, J.J. Brosens, J.J. Kim, Mol.Endocrinol.21 (2007) 2334.
[21]谢萍,李璐,张令强,等.微管结合蛋白NuSAP1的组织表达谱分析.军事医学科学院院刊,2010,34(6)：509-12.
[22]Chou HY, Wang TH, Lee SC, et al. Phosphorylation of NuSAP1 by Cdkl regulates its interaction with microtubules in mitosis[J].Cell Cycle. 2011,10(23):4083-4089.
[23]T. Fujiwara, H. Harigae, S. Takahashi, K. Furuyama, O. Nakajima, J. Sun, K. Igarashi,M. Yamamoto, S. Sassa, M. Kaku, T. Sasaki, Biochem. Biophys. Res. Commun.340(2006)105.
[24]S.K. Marie, O.K. Okamoto, M. Uno, A.P.G.Hasegawa, S.M. Oba-Shinjo, T. Cohen, A.A.Camargo, A. Kosoy, C.G Carlotti, S. Toledo, C.A. Moreira-Filho, M.A. Zago, A.J.Simpson, O.L. Caballero, Int. J. Cancer 122 (2008) 807.
[25]R. Satow, M. Shitashige, Y. Kanai, F. Takeshita, H. Ojima, T. Jigami, K. Honda, T.Kosuge, T. Ochiya, S. Hirohashi, T. Yamada, Clin. Cancer Res.16 (2010)2518.
[26]D.M. Kokkinakis, X. Liu, R.D. Neuner, Mol. Cancer Ther.4 (2005) 1338.
[27]B. Ryu, D.S. Kim, A.M. DeLuca, R.M. Alani, PLoS ONE 2 (2007) e594.
[28]D. Bogunovic, D.W. O'Neill, I. Belitskaya-Levy, V. Vacic, Y. Yu, S. Adams, F.Darvishian, R. Berman, R. Shapiro, A.C. Pavlick, S. Lonardi, J. Zavadil, I. Osman, N.Bhardwaj, Proc. Natl Acad. Sci. USA 106 (2009) 20429.
[29]D. Chen, A. Nasir, A. Culhane, C. Venkataramu, W. Fulp, R. Rubio, T. Wang, D.Agrawal, S. McCarthy, M. Gruidl, G Bloom, T. Anderson, J. White, J. Quackenbush,T. Yeatman, Breast Cancer Res. Treat.119 (2010) 335.
[30]M. Lauss, A. Kriegner, K. Vierlinger, I. Visne, A. Yildiz, E. Dilaveroglu, C.Noehammer, Breast Cancer Res. Treat.110 (2008) 235.
[31]J. Nie, H. Wang, F. He, H. Huang, Protein Cell.1 (2010) 259.
[32]F.M. Recchia, L. Xu, J.S. Penn, B. Boone, P.J. Dexheimer, Investig. Ophthalmol. Vis.Sci.51 (2010) 1098.
[33]B. Vogelstein, S. Sur, C. Prives, Nature Education.3 (2010) 6.
[34]S.W. Lowe, S. Bodis, A. McClatchey, L. Remington, H.E. Ruley, D.E. Fisher, D.E.Housman, T. Jacks, Science 266 (1994) 807.
[35]G.Cario, A. Fetz, C. Bretscher, A. Moricke, A. Schrauder, M. Stanulla, M. Schrappe,Ann. Hematol.87 (2008) 709.